To explore the standardization of the in vitro Nb2-11 cell proliferation bioassay for recombinant human growth hormone (rhGH), we first tested the biological activity of 2 batches of rhGH drug substances, 3 batches of rhGH for injection, and 24 batches of rhGH injections, based on which we proposed the experimental effective criteria. Furthermore, we conducted methodological validation in accordance with General Rule 9401 of the 2020 edition of the Pharmacopoeia of the People's Republic of China. Subsequently, the inter-laboratory consistency, intra-laboratory and inter-laboratory precision of seven labs were studied by determining the in vitro bioactivity of six batches of rhGH products and two batches of rhGH drug substances from four different manufacturers using this method. The consistency of the in vitro and in vivo bioassays was assessed by evaluating 28 batches of rhGH drug substances with both methods. Finally, multiple batches of rhGH samples with different expiration dates were tested to determine the standard limits of the bioassay. The results of this collaborative study indicate that the Nb2-11 cell bioassay exhibits good intra- and inter-laboratory reproducibility, with a 100% pass rate for the experimental effective criteria. The Nb2-11 cell bioassay had high consistency with in vivo animal bioassay. Animal experiments were approved by the Experimental Animal Welfare Ethics Committee of China National Institute of Food and Drug Control (approval number: NIFDC (Fu) No. 2024 (B) 004). This method is applicable to rhGH drug substances and products from different manufacturers. Collectively, the Nb2-11 cell bioassay can be used as a standardized method for determining the biological activity of rhGH products, replacing in vivo animal methods for quality control and release testing.

Transient receptor potential vanilloid (TRPV), an important class of non-selective cation channels, is closely associated with a variety of physiological and pathological processes. Pungent flavour is one of the important flavours in the doctrine of five flavours in traditional Chinese medicine, which has the functions of relieving the epidermis, moving the Qi, activating the blood and so on, which has a variety of main treatments. Modern research has shown that the active ingredients in numerous pungent Chinese medicine have the ability to modulate TRPV channels. The purpose of paper is to explore the biological characteristics of TRPV channels, the modern research process of pungent Chinese medicine and its mechanism in inflammation, pain and other pathological processes. This paper discusses the research progress of the pharmacological action of pungent Chinese medicine based on TRPV channel, in order to reveal the scientific connotation of spicy traditional Chinese medicine, and also provide new research ideas and models for the modern research of Chinese medicine.

In this thesis, we propose to establish a phage display method for screening adalimumab (ADM)-specific binding peptide to provide a particular element of recognition for clinical studies of ADM, and to successfully establish an indirect enzyme-linked immunosorbent assay (i-ELISA) based on this peptide for clinical detection of ADM. With ADM as the target molecule, five rounds of peptide elution were performed using phage display technology by putting it into the M13 linear dodecapeptide library. By measuring the titer of each round of eluate, it was found that the recovery rate increased from 7.28×10^-6 to 1.55×10^-3, indicating that the peptides binding to ADM in the eluate were continuously enriched, and the enrichment effect was better; the fifth round of screening amplicons were sequenced, and it was found that five peptide sequences appeared with high frequency, which were considered to be potentially able to bind specifically to ADM and were synthesised; the specific binding ability of the five peptide sequences to ADM was verified and identified by surface plasmon resonance (SPR) and ELISA experiments, respectively, and the results showed that the first peptide sequence (Pep1) and the second peptide sequence (Pep2) showed specific binding to ADM, with the affinity constants (K_D) of 7.91×10^-5 mol·L^-1 and 1.67×10^-5 mol·L^-1, respectively, and of which the affinity constant of Pep1 with ADM isotype antibody IgG was 1.35×10^-4 mol·L^-1, which was one order of magnitude lower than that with ADM, and thus Pep1 was determined to be a specific binding peptide for ADM. Based on Pep1, an indirect ELISA method for the analysis of the antibody-drug ADM was successfully established. With the increasing concentration of ADM the OD₄₅₀ value showed a regular change, and the method can be used for clinical ADM blood concentration monitoring. In this study, we obtained a peptide that can specifically bind to ADM, and this peptide sequence can be used as a specific recognition element for ADM, which not only enables the monitoring of ADM blood concentration, but also provides an effective tool for the accurate qualitative and quantitative quantification of ADM in vivo and ex vivo, and at the same time provides a new strategy and idea for the discovery of recognition elements of other monoclonal antibody drugs.

The critical relative humidity (CRH) is an important indicator for measuring the hygroscopicity of solid drugs. The intrinsic mechanism of rapid moisture absorption of water-soluble drugs under CRH is not fully understood, and we conducted preliminary exploration on this topic. Firstly, the newly developed model tablet moisture absorption method and the traditional powder moisture absorption method were employed to determine the CRH values, an equivalence test between the two methods was conducted by a two-tailed t test. Then a conductivity meter was employed to measure the conductivity values of drug model tablets under different relative humidity (RH) conditions. As we know, anhydrous copper sulfate is a grayish white powder, which is easy to turn blue after absorbing water, based on this, this salt was selected as a model drug, and its CRH value was measured. Furthermore, anhydrous copper sulfate was compressed into tablets, then placed under different RH conditions. A series of photos were taken to record the formation process of the liquid film. The reason for the inflection point of the moisture absorption equilibrium curve was postulated based on changes in conductivity and color. The results showed that the CRH values of sucrose powders and tablets were 85.05% and 85.01%, respectively; and the CRH values of sodium citrate powders and tablets were 77.14% and 76.93% individually; the results of the two-tailed t test indicated that the two methods are equivalent. At RH of 75%, the conductivity of sodium citrate changed slightly, while at RH of 79% greater than the CRH, the conductivity underwent a sudden change. Sucrose and anhydrous copper sulfate showed a similar trend of change. In addition, the process of blue dots of anhydrous copper sulfate changing from discrete to continuous was successfully captured during the observation of moisture absorption. In summary, the aforementioned results suggest that the mechanism of the rising rapidly in moisture absorption curve of water-soluble drugs at CRH is probably attributed to the formation of a continuous aqueous "liquid film" on the drug surface during the process of moisture absorption.

The main chemical components of Gandoufumu Decoction (GDFMD) were analyzed and identified using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS^E). We utilized a ZORBAX RRHD Eclipse Plus C₁₈ column (2.1 mm × 100 mm, 1.8 μm) with a gradient elution of 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.2 mL·min^-1 and a column temperature of 35 ℃. The mass spectrometry was analyzed using an electrospray ionization source (ESI) in positive and negative ion modes. The UPLC-Q-TOF/MS^E was used to detect and analyze the complex mass spectra and the chemical composition of the GDFMD. The results show that 102 compounds were identified in GDFMD, 26 flavonoids, 22 terpenoids, 19 saponins, 10 phenylpropanoids, and 25 other components. The established qualitative method can rapidly and efficiently identify the chemical components of GDFMD, providing a scientific basis for the quality evaluation and clinical application of GDFMD.

Arjunic acid (AR), a main bioactive triterpenoid isolated from acorns, has been reported to exert pronounced anti-inflammatory activities. However, its anti-inflammatory mechanisms have not been elucidated. In this study, the model of lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells were established to investigate the anti-inflammatory activity of AR. The potent targets and signaling pathway of AR for the treatment of inflammation-related disease were predicted based on network pharmacology. Furthermore, the expression of pro-inflammatory cytokines and mediators was determined by Griess assay, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, and Western blot. The protein expression of NF-κB, MAPK, Nrf2/HO-1, PI3K/Akt/mTOR, and autophagy signaling pathways were gauged by Western blot. As the result, in the inflammatory model of LPS-induced RAW264.7 cells, AR could significantly inhibit the expression of pro-inflammatory cytokines and mediators, suppress the phosphorylation and translocation of NF-κB, and downregulate the phosphorylation of JNK/ERK signaling pathways. AR also inhibited ROS production and activated the Nrf2/HO-1 signaling pathway by degrading Keap1. Furthermore, AR activated autophagic flux by inhibiting the PI3K/Akt/mTOR signaling pathway. Collectively, AR was a potential natural product for the treatment of inflammation-related diseases.

The purpose of this study is not only to investigate the effects of Angelica sinensis polysaccharide (ASP) as a potential vaccine adjuvant on immune activation and cytokine release in RAW264.7 macrophages, but also to elucidate its underlying involved signaling mechanisms. Cell viability was evaluated by the CCK-8 assay. Flow cytometry was used to analyze the influence of ASP at five distinct concentration gradients on the expression of cluster of differentiation (CD) 80, CD86, and major histocompatibility complex Ⅱ (MHC Ⅱ) on RAW264.7 cell surfaces. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA) method. Molecular techniques, including quantitative polymerase chain reaction (qPCR) were utilized to assess the mRNA expression levels of Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-TNF receptor associated factor 6 (TRAF6)-nuclear factor kappa-B (NF-κB) signaling pathway. The levels of TRAF6, and the phosphorylation levels of IκB kinase (IKK) and p65 proteins were detected by Western blot. The results show that ASP at varying concentrations promote the proliferation of RAW264.7 cells without cytotoxicity. Surface molecules CD80, CD86, and MHC Ⅱ on RAW264.7 cells showed statistically significant up-regulation in response to ASP compared to the blank control (P < 0.05), with a dose-dependent effect within an optimal range. Furthermore, ASP also elevated cytokines IL-6 and TNF-α secretion levels by RAW264.7 cells compared to the normal control (P < 0.05), exhibiting a dose-response relationship within a specific concentration span. The qPCR results indicated that ASP groups at different concentrations all led to upregulation of mRNA expression levels of TLR4, MyD88, TRAF6, and NF-κB signaling pathway. The expression levels of TRAF6, p-IKK and p-p65 were increased by different concentrations of ASP. The TLR4 inhibitor TAK-242 significantly reduced the secretion of cytokines induced by APS (P < 0.05). This study highlights the immunostimulatory properties of ASP, emphasizing its potential as a vaccine adjuvant. By significantly enhancing the expression of co-stimulatory molecules and cytokines via the TLR4-MyD88-TRAF6-NF-κB signaling pathway, ASP offers a promising approach for modulating immune responses.

In the study, we employed an untargeted metabolomic approach in conjunction with 16S rRNA high-throughput sequencing to identify potential antidepressant active effectors mediated by gut-flora among geniposides. Firstly, the rat depressed model was constructed using chronic unpredictable mild stress stimulation (CUMS) combined with orphaned model. Then the cecal contents of the rats were analyzed by untargeted metabolomics using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) in order to identify differential metabolites. The intestinal genera exhibiting significant differences in the cecal contents were identified through 16S rRNA high-throughput sequencing, and correlation analysis was subsequently conducted between the differential metabolites and the intestinal genera. The comparison of the metabolic profiles between the normal control group, the depression model group, and the geniposide treatment group revealed that 147 metabolites were down-regulated and 381 metabolites were up-regulated in the model group compared with the normal group. Furthermore, 212 metabolites were up-regulated and 288 metabolites were down-regulated in the geniposide treatment group compared with the model group. Subsequently, the combination of the screening conditions of FC (fold change) > 2, P < 0.05 and VIP (variable importance in the projection) > 1, secondary mapping and database comparison led to the identification of 55 metabolites with significant differences among the three groups. The results of the metabolic pathway enrichment analysis indicated that the identified differential metabolites were primarily involved in five metabolic pathways, namely tryptophan metabolism, arginine biosynthesis, phenylalanine biosynthesis with tyrosine and tryptophan, phenylalanine metabolism, and arginine metabolism with proline. The 16S rRNA sequencing results further indicated that Gardenia jasminoides extract may exert its antidepressant effects, thereby providing a new basis for the study of the gut-brain axis in the therapeutic strategy of depression. All animal experiments were conducted with the approval of the Biomedical Research Ethics Committee of the Naval Medical University.

The HIV-1 capsid protein (CA) is an effective target for antiviral drug discovery. Targeting the capsid assembly inhibitor peptide (CAI) binding pocket in the C-terminal domain of HIV-1 CA (CA CTD) can be used to screen for CA protein inhibitors. In this study, we found that the small molecule suramin targets HIV-1 CA by binding to this pocket, with an IC₅₀ value of 2.1 μmol·L^-1 for inhibiting the CA CTD-CAI interaction, based on homogeneous time-resolved fluorescence (HTRF) technology. Bio-layer interferometry (BLI) experiments demonstrated that suramin binds directly to CA, with a binding affinity to CA hexamer (K_D = 248 nmol·L^-1) higher than to CA monomer (K_D = 227 μmol·L^-1). In addition, in vitro CA protein assembly assays showed that suramin promotes disordered multimerization of CA protein. Binding model analysis revealed that suramin binds to the interface between adjacent CA monomers in the CA hexamer through the N57, Q63, Q67 and Y169 residues, thereby enhancing CA multimerization. In summary, suramin is a CA protein inhibitor that targets the CA hexamer and can serve as a starting point for the discovery of new CA inhibitors.

Colorectal cancer is a major cancer threatening the life and health of people worldwide. With the transformation of modern medical models, treatment strategies represented by traditional Chinese medicine (TCM) with multiple targets have become a new research direction in the treatment of colorectal cancer. In the study of TCM, in addition to deconstructing the components and targets of TCM based on reductionism, more attention should be paid to the holistic concept in the basic theories of TCM, analyzing and understanding the mechanism of action of TCM treatment from a holistic perspective. TCM compound prescriptions, as a collection of single herbs, are composed of various monomers. Viewing TCM from different levels and considering different herbs and monomers as a whole helps us better understand TCM. At the same time, TCM has the characteristic of multiple targets. In addition to directly acting on tumor cells themselves, it can also affect the intestinal flora to improve the tumor microenvironment, treating tumors in an all-round and multi-dimensional way, regulating the physical functions of tumor patients from a holistic perspective, and comprehensively improving the health status of the individual. This review aims to systematically elaborate on the efficacy and multi-target mechanism of TCM in the treatment of colorectal cancer from three levels: TCM compound prescriptions, single herbs, and TCM monomers, based on clinical trials and preclinical research results. Meanwhile, this article will combine systems biology with the holistic view of TCM to conduct an in-depth analysis of related research on TCM treatment of colorectal cancer.